Confocal Raman Spectroscopy for Dermatological Studies

ABSTRACT

Use of Confocal Raman Spectroscopy (CRS) for dermatological studies, including a method for determining the thickness of the Stratum Corneum (SC) on a test area of the skin, and to a method for quantifying the effectiveness of a skin care composition. The methods can be carried in vitro (either artificial skin or a sample of skin) or in vivo (directly on the human skin of a person).

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of application Ser. No. 11/510,898,filed Aug. 28, 2006, which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the use of Confocal Raman Spectroscopy(CRS) for dermatological studies. In particular, the invention relatesto a method for determining the thickness of the Stratum Corneum (SC) ona test area of the skin, and to a method for quantifying theeffectiveness of a skin care composition. The methods of the inventioncan be carried in vitro (either artificial skin or a sample of skin) orin vivo (directly on the human skin of a person).

BACKGROUND OF THE INVENTION

Skin is composed of two main layers, the dermis and epidermis, which arein turn composed of sub-layers. The surface of the skin is the outermostlayer of the epidermis and is called the Stratum Corneum. It is composedmainly of dead cells that lack nuclei and contain keratin, a proteinthat helps keep the skin hydrated by preventing water evaporation. Inaddition, these cells can also absorb water.

The standard method for measuring skin hydration in the Stratum Corneumis to measure a change in the electrical properties of the skin(specifically the capacitance), which is related to the degree ofhydration. The apparatus commonly used for this measurement is called aCorneometer® (available from Courage & Khazaka).

Water has a very high dielectric constant, closely followed byglycerine, both higher than the dry skin values. Therefore it is assumedthat an increase in Corneometer values can be equated with an increasein moisturization (mainly due to a water effect, however contributionsfrom glycerine cannot be ruled out). However the scale is not linear,and therefore the technique is not quantitative. Also the depth overwhich the data is collected is poorly defined. Although the depth rangeis believed to cover the entire Stratum Corneum it may well cover aportion of the viable epidermis as well. With this technique it is alsonot possible to get information on the distribution of water within thestratum corneum, with only a single number being generated per reading.In addition, alternative ways of making such measurements must often bemade by invasive methods.

The present invention uses a different technique to measure skinmoisturization, based on Confocal Raman Spectroscopy. Raman spectroscopyis the measurement of the wavelength and intensity of inelasticallyscattered light from molecules. Raman scattered light occurs atwavelengths that are shifted from the incident light by the energies ofmolecular vibrations. The mechanism of Raman scattering is differentfrom that of infrared absorption, and Raman and IR spectra providecomplementary information. For further background information on Ramanspectroscopy, see for example “Fundamentals of Molecular Spectroscopy”,C. N. Banwell, McGraw Hill, 1983.

Two of the major advantages of Raman Spectroscopy are the nondestructive nature of this technique and the virtually no need forsample preparation that it requires, which may provide significant costand time savings.

Confocal optics relate to the illumination of a sample with adiffraction limited spot such that the illuminating spot is imaged on anideally point-like detector, the point-like detector being realised withan adjustable pinhole called ‘confocal hole’ in front of the realdetector (entrance slit). An advantage of confocal sampling is theability to separate the signal from each layer of a layered sample. Inthe case of skin measurement, Confocal Raman Microspectroscopy allows tomeasure the property of skin as a function of depth.

It has been recently proposed to apply Raman Microspectroscopy to theanalytical determination of skin moisturization, see:

-   -   “Automated depth-scanning Confocal Raman microspectrometer for        rapid in vivo determination of water concentration of water        concentration profile in human skin”, P. J. Caspers, G. W.        Lucassen, H. A. Bruining and G. J. Puppels, J. Raman Spectrosc.        31, 813-818 (2000);    -   “In vivo Confocal Raman Microspectroscopy of the Skin:        Noninvasive Determination of Molecular Concentration        Profiles”, P. J. Caspers, G. W. Lucassen, E. A. Carter, H. A.        Bruining and G. J. Puppets. The Journal of Investigative        Dermatology, Vol. 133, No. 3 March 2001, 434-442;    -   “Confocal Raman Microscopy for Cosmetic Applications”, published        in “Raman Update”, a publication by HORIBA Jobin Yvon, Winter        Edition 2005.

Although some of the above mentioned documents have recognized theusefulness of Confocal Raman Spectroscopy to study the profile ofhydration within the Stratum Corneum as a function of depth, as well asa function of time (for example pre- and after-application of a skinmoisturization composition), the inventors have made the surprisingdiscovery that, in some cases, the data measured by this advancedtechnique leads to incoherent results, as is discussed below.

Whilst making measurements of skin hydration of the Stratum Corneum, theinventors found that, unexpectedly, the hydration value as measured byConfocal Raman Spectroscopy at a specific depth within the StratumCorneum decreased after the application of certain skin care hydrationproducts. These results contradicted visual examination of the surfaceof the area of the skin tested as well as Corneometer measurements,which indicated an overall increase in skin hydration at the surface ofthe skin area. After further experimentation and insight, and whilst notwishing to be bound by theory, the inventors have come to the conclusionthat certain skin care products can be so beneficial to the health ofthe skin that they increase the absolute depth of the Stratum Corneum ofthe users.

As will be discussed in details further below, the inventors have thenfound that in order to determine the effectiveness of skin carecompositions a crucial factor that was previously overlooked is theevolution of the thickness of the Stratum Corneum. In addition to thisinsight, the inventors have developed a method for determining thethickness of the Stratum Corneum using Confocal Raman Spectroscopy.Traditionally the thickness of the Stratum Corneum has been measured byusing biopsies, however this is an inherently destructive process,requiring removal of a section of flesh from the body.

SUMMARY OF THE INVENTION

A first aspect of the invention is a method for determining thethickness of the Stratum Corneum on a test area of skin using ConfocalRaman Spectroscopy. The method comprises the steps of:

-   -   (a) measuring the concentration profile of a Raman-active        material (for example water) as a function of depth within the        test area using Confocal Raman Spectroscopy; then    -   (b) processing the data obtained to determine the thickness of        the Stratum Corneum.

A second aspect of the invention is for a method for determining theeffectiveness of a skin care composition. The method uses the thicknessof the Stratum Corneum (preferably as determined by the method of thefirst aspect of the invention) before and after application of the skincare composition as a parameter to quantify the effectiveness of thecomposition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a typical high wavenumber spectrum of hydrated skin and therelevant areas measured when determining the % hydration level.

FIG. 2 shows a typical Confocal Raman Spectroscopy profile of the watercontent of skin as a function of depth. In this example, a typical waterprofile showing the surface of the stratum corneum at depth=0 with20-30% hydration, rising to the 60-70% hydration within the body at adepth of approximately 20 μm for forearm skin. The line of best fitthrough the data points has been fit using a Weibull growth curve model.

FIG. 3 shows the hydration profile before and after application ofpetrolatum.

FIG. 4 shows the Raman hydration profiles (averaged across all subjectsby treatment) before product application at the start of a study tocompare two treatment regimes (‘a’ and ‘b’).

FIG. 5. shows change from baseline in % hydration measured at a fixeddepth (10 μm) below the surface of the SC for both treatment regimes(‘a’ and ‘b’) during 2 weeks of product application and after 1 weeksregression.

FIG. 6 shows the Raman hydration profiles (averaged across all subjectsby treatment) after 2 weeks of product application for the treatmentregimes ‘a’ and ‘b’.

FIG. 7 shows the Raman hydration profiles (averaged across all subjectsby treatment) after 1 week regression for the treatment regimes.

FIG. 8 shows the change from baseline in total area under the curve fromthe surface of the SC to the leveling off point (determined from theRaman profile) during 2 weeks of product application and 1 weekregression for 2 treatment regimes.

DETAILED DESCRIPTION OF THE INVENTION Confocal Raman Spectroscopy

Confocal Raman Spectroscopy uses a microscope system to focus laserlight to a point. The light at the point of focus is of high intensitywhich is where the Raman signal is generated from. By altering theposition of the microscope objective lens relative to the sample to beanalysed, and when the sample is not completely opaque to the incidentlaser light the interior of the sample can be examined. By moving theobjective lens in small increments a profile of Raman spectra as afunction of depth can be produced. The Raman spectra contain peakscorresponding to the different functional groups of the chemicalspresent within the sample. The locations of these peaks are determinedby the precise chemical structure of the components. Once the peaklocations for different components of the sample are known, ratioing ofone component to the others present can be carried out. For instance, %water in skin can be calculated by ratioing the amount of water andprotein as calculated from the areas under the curves in the part of thespectra corresponding to water and protein respectively, and applying aproportionality constant (as detailed below).

Any suitable commercially available CRS equipment can be used. Forexample the inventors used a River Diagnostics Model 3510 Confocal RamanMicrospectroscopy system (software version—RiverIcon v.1). This has beendesigned for use as an in-vivo, non invasive skin analysis device,enabling qualitative and semi-quantitative analysis of molecularconcentrations and concentration profiles within the skin. The systemincorporates a CCD detector combined with a microscope objective lens toenable focusing of the laser light into the skin and collection of thereturning signal. 2 lasers are used—a 671 nm red laser for waterprofiling (operating in the high wavenumber region from 2500-4000 cm⁻¹),and a 785 nm near IR laser for low wavenumber region and naturalmoisturising factor (NMF) measurement. Profiles in the high wavenumberregion may be measured using 1s acquisition times per spectra, and inthe fingerprint using 10s acquisitions per spectra. Typically 2 or 3 μmspacings between spectra may be used. The top few hundred microns of theskin is transparent to the light from both the 671 nm and 785 nmallowing profiling within the SC using this arrangement.

The points forming the hydration profile as function of depth arederived by the software using the Raman spectra acquired for each depthvalue. The software may use the calculation method as outlined in PeterCaspers Ph.D. Thesis (‘In-vivo skin characterization by confocal Ramanspectroscopy’, 2003, Erasmus University, Rotterdam, the Netherlands).For example, as explained in this thesis, the area between 3350-3550cm⁻¹ may be integrated for the water band [water], and 2910-2966 cm⁻¹for the protein band [protein] (a sample spectra showing the areasmeasured for water and protein is given in FIG. 1).

The percentage hydration may then be calculated for each depth with thisformula:

% hydration=[water]/([water]+r.[protein])

wherein r is a proportionality constant,

This procedure is carried out automatically at each point of the spectraby the associated RiverIcon software and results in the formation of ahydration profile (see FIG. 2). A similar process may be followed whenlooking at different active species (for example vitamins, and aminoacids), where a principal component analysis using well defined peaklocations is used to calculate a profile for each of the ingredients ofinterest. Again this is carried out within the standard softwareprovided with the equipment and the data outputted in the form of aprofile for the ingredient of interest as a function of depth.

Raman Active Materials Present in Skin

In principle, anything which is Raman active can be measured within theskin using this technique. For a specific vibrational mode to be Ramanactive, there must be a change in the polarizability of the moleculecaused by the vibration. It has already been shown in the literature(papers by Puppels and Caspers from River Diagnostics) that water andthe amino acids which make up natural moisturizing factors (NMF) can beanalyzed within the skin, along with cholesterol, lactic acid, andkeratin. Due to the complex structure of most ingredients of interestwithin skin care formulations there will normally be some vibrationsassociated with any given molecule of interest which will be Ramanactive. In order for the molecules of interest to be measured in a Ramanprofile they must fulfill two criteria, i) that they have peaks whichare sufficiently distinct from other components within the skin, and ii)that the ingredient is present in sufficient quantity to be detected.The absolute intensity of the peaks in a spectrum will be determined byhow strong the change in polarizability is, and will vary from compoundto compound. Peak location within the spectrum is determined by thefunctional groups present within the molecule.

Treatment of the Data

The data points gathered are processed to be more readily usable. Datapoints that make up each profile may be saved as a tab delimited textfile and imported into a suitable mathematical software, for exampleMatlab. In the exemplary system used, up to 8 profiles for any givensite may be imported. The dataset (containing all profiles) may then betreated as a cloud of points through which a line of best fit is put.The mathematical model for the line of best fit may be based on theWeibull model, although different models may be used (e.g. polynomialregression).

The Weibull distribution is widely used in reliability and life (failurerate) data analysis and as a biological growth model. The equation forthe Weibull model used here is given below.

y=a−(a−b)*exp(−(x/c)̂d)

Where a, b, c and d are variables determined during the optimization ofthe line of best fit by the mathematical software. A line of best fitbased upon this model is fitted to the dataset (see FIG. 2), and usingthis equation different parameters of the skin can be determined (forexample, bottom of the stratum corneum (SC), complete area under thecurve from the surface to the base of the SC).

Determination of the Thickness of the Stratum Corneum

During the calculation of the line of best fit through the dataset, thedetermination of the leveling off point of the curve is also carriedout. The leveling off point is determined using a gradient threshold onthe Weibull model. A value for the gradient threshold may be set by theoperator during data analysis. The leveling off point is taken as wherethe slope on the modeled curve matches the threshold set. This levelingoff point corresponds to where the water rich living tissue of theepidermis meets the SC, i.e. the bottom of the SC.

When analyzing an entire study, a subset of the data is chosen at randomand analysed using different gradient threshold values, the operatorthen determines the most accurate fit for the leveling off point anduses the corresponding gradient threshold value for analysis of theentire study. The need for operator choice for the gradient thresholdarises from a number of factors. For example the skin on different bodyparts has inherently different water profiles, also the skin's naturalhydration state is strongly influence by the time of year and associatedweather conditions. It should be emphasized though, that once a valuefor the gradient threshold has been derived for the small subset of datafrom the entire study, that value is normally used for the entireanalysis. Typical values for the gradient threshold on volar forearmskin are between 0.4 and 1.0, and this range may be used as a startingpoint when determining the appropriate value. An example data set fittedwith the Weibull model is shown in FIG. 2. It is also be possible to useother mathematical operations to determine the leveling off point, suchas the point at which the % hydration reaches a fixed percentage of theupper asymptote of the Weibull model. As with the use of the gradientthreshold, this provides a route to determining the location of thebottom of the SC.

Determination of the Effectiveness of a Skin Care Composition

The effectiveness of a skin care composition is normally expressed asthe change of a certain skin quality between the beginning and the endof the study. Confocal Raman Spectroscopy may be used to quantify thechange of concentration of a Raman active material within the skin, andtherefore may be used to determine the effectiveness of a skin carecomposition when a Raman active material can be linked the effect of thecomposition studied. For example, a change in skin hydration, which canbe linked to the concentration of water within the skin, can be measuredusing the CRS technique because water is a Raman active material.Similarly, any Raman active materials that can be linked to theeffectiveness of a skin care composition (Natural Moisturizing Factors(NMF), Vitamins, etc. . . . ) may also be used to quantify itseffectiveness.

The term “skin care composition” as used herein refers to a product thatis intended to have an effect on skin. The term includes cosmeticproduct, whose purpose to improve the appearance of skin, as well astherapeutic treatment, whose purpose is to prevent or treat a skindisease (these terms are not mutually exclusive). Non-limiting examplesof skin-care products include leave-on products (e.g. moisturizingcreams, self-tanning products, tinted moisturizers, powders,foundations, conditioning wipes, etc. . . . ) and rinse-off products(shower gels, in-shower moisturizers, foaming wipes, etc. . . . ). Alsoincluded are products which are not directly applied on the skin, e.g.nutraceuticals which are ingested by the user.

The inventors have now found that for effectively quantifying theeffectiveness of a skin care composition using Confocal RamanSpectroscopy, it is important to take into account the change ofthickness of the SC during the study. Without wishing to being bound bytheory, this may be because changes in the hydration state of the skinalter its thickness, or that certain skin actives (e.g. Niacinamide) mayincrease skin cells proliferation. Therefore it may not be appropriateto compare values obtained at the beginning and the end of the study ata constant depth (e.g. 10 μm).

For actives delivered from the composition, it is normally important toknow depth of penetration and % concentration as a function of depth. Assuch it is important to reference any change in the quantity of a Ramanactive material % hydration changes to % of the way through the SC. Alsoas the thickness of the SC may have changed, the parameter of total areaunder the curve from the surface to the bottom of the SC becomesimportant as a total hydration measure. Wrongly considering that the SCto be fixed in thickness throughout the study may lead to incorrectinterpretation of the data.

The thickness of the SC at the beginning and then at the end of thestudy may be determined using the method described above which employs aCRS technique. Measuring the water concentration profile using CRS andprocessing the data obtained was found to be a good way to determine SCthickness. If the effect to be measured is skin hydration, then only onemeasure of the concentration profile at the beginning and at the end ofthe study needs to be performed, because the data generated fordetermining the SC thickness can also be used to determine the watercontent of the SC.

There are different ways to express the effectiveness of a skin carecomposition using the data generated by CRS and the SC thickness, ofwhich two preferred examples are outlined here:

-   -   a specific relative depth of the SC (e.g. half-way) may be        selected, and the amounts of Raman-active substance (e.g. water)        linked to the effect of the skin care composition (e.g. skin        hydration) to be determined at this relative depth at the        beginning and at the end of the study may be compared;    -   another way to express the effectiveness of the composition is        to measure the area under the curve (integrating), between the        skin surface and the end of SC (e.g. as determined by CRS, as        described above). Dividing the value obtained for the surface        area at the end of the treatment by the value obtained for the        surface area at the beginning of the treatment gives a measure        of the effectiveness of the composition in %. This method of        quantifying the effect also works well for quantifying skin        hydration. Also the total area under the curve for individual        NMF's could be linked to health of the skin (as NMF's are        beneficial to the water holding capability of the SC and are        readily washed out).

EXAMPLE 1 Single Variable Analysis of Hydration Levels Within theSkin—Petrolatum Occlusion

To demonstrate the effects of a single variable on skin hydration, a setof baseline spectra were recorded (the site to be used was dry wiped toremove surface sebum before the measurements were taken). Petrolatum wasthen applied to the same area of the forearm 4 times over a 24 hourperiod with the aim of promoting skin hydration via occlusion. After 24hours the site was dry wiped to remove any surface contamination and afurther set of profiles collected (FIG. 3). This shows how the hydrationlevel near the surface of the SC has increased due to occlusion (x=0 to5 μm). Also the total area under the curve from the surface to thebottom of the SC has increased from 697 to 764—an increase ofapproximately 10%.

EXAMPLE 2 Effect of Moisturing Products

In this study, two commercial moisturizing treatments (‘a’—Olay® Quench,and ‘b’—Jergens® Ultra Healing) were used. After an initial baselinereading product, the products were applied for 2 weeks followed by a 1week regression period during which no product was applied to the sitesexamined. Product application was 2 μl cm⁻², twice daily, over sites onthe volar forearms of 15 panelists. Panelists did not use moisturizingproducts other than those provided by the study organizers on theirforearms over the entire course of the study. The baseline profile forskin hydration at the beginning of the study (no products applied) isshown on FIG. 4. As shown, the baseline profile for both sites ‘a’ and‘b’ were identical.

The change in % hydration for the two moisturizing treatments ‘a’ and atthe fixed depth of 10 μm data beneath the surface of the SC is given inFIG. 5. Looking at the data in FIG. 5, treatment ‘a’ appears to beresulting in a dehydration of the SC at 2 weeks usage and after 1 weekregression.

However if the shape of the profile at each of these time points isexamined, there is a clear difference for treatment ‘a’ after 2 weeksusage and 1 week regression. FIGS. 6 and 7 show a change in the skinthickness as the leveling off points for treatment ‘a’ and ‘b’ aredifferent (where as at the start of the study—the baseline reading, FIG.4, shows that the skin at all the sites is equivalent as the levelingoff points are coincident). Therefore measurement only of % hydration ata single depth beneath the surface of the SC is misleading, as treatment‘a’ would have appeared to have lowered in % hydration at a fixed depth.

Use of the total area under the curve calculated by taking into accountthe leveling off point, i.e. total hydration level within the SC isshown in FIG. 8. This shows a clear and statistically valid (p<0.05)increase in the total hydration within the skin (i.e. total hydrationcontent within the SC) for treatment ‘a’ which was not observed byexamining the % hydration at a fixed depth (the change shown correspondsto approximately a 10% increase in area under the curve is seen forproduct ‘a’). All the data generated for this study was analysed using agradient threshold of 0.5.

In addition to the obvious interest of the invention for dermatologicstudies, it is envisaged that the invention may also be useful forgenerating advertising claim support for commercial skin care products.Although the invention will normally be used in a lab, it is alsoenvisaged that the apparatus may be made mobile so as to be used in“road show” event, for example in supermarkets.

All documents cited in the Detailed Description of the Invention are, inrelevant part, incorporated herein by reference; the citation of anydocument is not to be construed as an admission that it is prior artwith respect to the present invention. To the extent that any meaning ordefinition of a term in this written document conflicts with any meaningor definition of the term in a document incorporated by reference, themeaning or definition assigned to the term in this written documentshall govern.

While particular embodiments of the present invention have beenillustrated and described, it would be obvious to those skilled in theart that various other changes and modifications can be made withoutdeparting from the spirit and scope of the invention. It is thereforeintended to cover in the appended claims all such changes andmodifications that are within the scope of this invention.

1. A method for determining the thickness of the Stratum Corneum on atest area of skin comprising the steps of: (a) measuring theconcentration profile of a Raman-active material as a function of depthwithin the test area using Confocal Raman Spectroscopy; then (b)processing, using a computer, the data obtained to determine thethickness of the Stratum Corneum.
 2. The method of claim 1, wherein thedata is processed using the Weibull algorithm.
 3. The method of claim 1,wherein the measurement is made in vivo.
 4. The method according toclaim 1, wherein the Raman-active material is selected from the groupconsisting of water, NMF, vitamins, ingredients from a personal carecomposition, and combinations thereof.
 5. The method according to claim4, wherein the Raman-active material is water.
 6. The method of claim 1,wherein the thickness of the Stratum Corneum is determined before andafter application of a skin care composition.
 7. The method of claim 6,wherein the skin care composition comprises at least one moisturizingagent.
 8. The method of claim 6, wherein the Raman-active substance islinked to the effectiveness of the skin care composition.
 9. The methodof claim 8, wherein the effectiveness of the skin care composition iscalculated by selecting a specific relative depth of the Stratum Corneumand comparing the amount of the Raman-active substance present at thespecific relative depth before and after the application of the skincare composition.